Mechanisms of neutrophil recruitment in septic lung injury

Sepsis is one of leading cause of death despite aggressive surgical intervention and antibiotic therapies. Excessive neutrophil recruitment is a major feature in early phase with immune system dysfunction at later phase. Lung is the most vulnerable, critical and sensitive organ during sepsis process. The aim of thesis was to prevent excessive neutrophil accumulation in lung parenchyma on one hand, and to reinforcement of immune system at later phase of sepsis on other hand. We hypothesized that CD44 may have a role in mediating pulmonary recruitment of neutrophils along with Rho GTPase subfamily in more than one way for enhancing neutrophil stiffness and migration. Peptides are new evolutionary compound with multifunctional effects, especially during infection and sepsis but potential therapeutic effect of them in polymicrobial sepsis remains elusive. Polymicrobial sepsis was induced by cecal ligation and puncture, purified monoclonal antibody against CD44, Rho kinase inhibitor (Y-27632) and Rac1 inhibitor (NSC23766). Specific TDPs, GKY20 and GKY25 were injected after procedure. Edema formation, bronchoalveolar accumulation of neutrophils, myeloperoxidase activity, and CXC chemokine in lung measured after CLP. We observed that sepsis triggered clear-cut lung damage characterized by edema formation, neutrophil infiltration, and increased levels of MIP-2 in the lung. We demonstrate that immunoneutralization with anti-CD44 reduce neutrophil activation and accumulation as well as edema formation and lung injury. Pretreatment with Y-27632 reduced the CLP-induced pulmonary injury and MPO activity as well as Mac-1 on neutrophils along with clear reduction in F-actin formation. Administration of NSC23766 markedly reduced CLP-triggered neutrophil infiltration, edema formation and tissue damage in the lung. Inhibition of Rac1 decreased CLP-induced neutrophil expression of Mac-1 and pulmonary formation of CXC chemokines. NSC23766 abolished the sepsis-evoked elevation of mRNA levels of CXC chemokines and TNF-α in alveolar macrophages. Moreover, TDPs maintain CD4 T-cells function in spleen by reducing T-cell apoptosis and clear reduction in sepsis-mediated T-regulatory production. TDPs abolished CLP- evoked HMGB1 and IL-6 production. Furthermore TDPs exerts clear cut bacterial clearance in the blood and spleen. Thus, this work show more details in neutrophil extravagation during sepsis. Our data may dig up the way for establishing more specific and effective treatments of sepsis

Targeting CD44 Expressed on Neutrophils Inhibits Lung Damage in Abdominal Sepsis.

Neutrophil infiltration is an insidious feature in septic lung injury, although the specific adhesive mechanisms regulating pulmonary recruitment of neutrophils in polymicrobial sepsis remain elusive. The aim of this present study was to define the role of CD44 in sepsis-induced neutrophil infiltration and lung damage. Mice were treated with a monoclonal antibody against CD44 before cecal ligation and puncture (CLP) induction. Edema formation, bronchoalveolar accumulation of neutrophils, myeloperoxidase activity, and macrophage inflammatory protein-2 (MIP-2) levels in the lung were determined after CLP. Expression of Mac-1 and CD44 on neutrophils was quantified by using flow cytometry. In separate experiments, fluorescent-labeled neutrophils co-incubated with an anti-CD44 antibody were adoptively transferred to CLP mice. CLP triggered clear-cut lung damage characterized by edema formation, neutrophil infiltration, and increased levels of MIP-2 in the lung. Notably, immunoneutralization of CD44 reduced CLP-induced pulmonary accumulation of neutrophils. In addition, functional inhibition of CD44 decreased CLP-induced lung damage and edema. However, formation of MIP-2 in the lung and neutrophil expression of Mac-1 were intact in septic mice pretreated with the anti-CD44 antibody. Adoptive transfer experiments revealed that neutrophil rather than lung CD44 mediates neutrophil accumulation in septic lung injury. Moreover, administration of hyaluronidase had no effect on CLP-induced neutrophil recruitment and tissue damage in the lung. Our data demonstrate that CD44 contributes to pulmonary infiltration of neutrophils and lung damage associated with abdominal sepsis. Thus, these novel findings suggest that CD44 may serve as a target to protect against lung injury in polymicrobial sepsis

Effect of changes in some beneficial elements and dangerous heavy metals in cow and sheep manure on human and animal health and the environment

Cow and sheep manure contains beneficial nutrients nitrogen (N) carbohydrates (CHO) and Organic matter (OM), and is therefore used as an organic fertilizer. However, the excessive use of manure can cause environmental problems and negatively affect human and animal health because these materials also contain large amounts of heavy metals, especially selenium (Se), nickel(Ni), cobalt(Co), tin(Sn), lead(Pb), and cadmium(Cd) To assess the potential risks due to increased concentrations of heavy metals in sheep and cow manure.The compost was collected from three different regions in Kirkuk, Diyala and Sulaymaniyah governorates in Iraq and Kurdistan Region where the results showed The content of cow and sheep manure of organic matter (OM), carbohydrates (CHO) and nitrogen (N) levels were normal, which are beneficial nutrients for soil and plants and used as organic fertilizer. The level of selenium, nickel and cobalt was higher in cow manure compared to sheep manure in all governorates. The level of lead and cadmiumwas higher in sheep manure in Diyala governorates compared to cow manure

Cooperative and antagonistic reactions of heavy metallic elements and its impact on health that are supplemented with selenium and zinc in Kurdi sheep

The purpose of present study was to determine the variations  in the levels of Selenium, Zinc, Iron, Copper, Molybdenum and Manganese in the serum of Kurdi sheep  and to compare the age difference in the levels of minerals between lambs and rams that are supplemented with Selenium and Zinc individually or their combination.It was on two experiences.The males of the Kurdi sheep from lambs and rams for four coefficients each as follows; First Treatment: (control) without adding, Second Treatment: added Selenium with a dose of 0.5 mg /kg fed,Third Treatment: added Zinc with a dose of 100 mg /kg fed, Fourth Treatment: added Selenium with Zinc with a dose of 0.5 + 100 mg/kg fed, then they were given gelatinous capsules daily for 60 days.Selenium and Zinc supplementation and their combination led to significant increase in the Selenium and Zinc concentration in the blood serum of lambs and Rams. Selenium, Zinc and their combinationsignificantly contributed to changes in the analysed minerals (Selenium, Zinc, Iron, Copper Molybdenum and Manganese)

Effect of Changes in Some Beneficial Elements and Dangerous Heavy Metals in Cow and Sheep Manure on Human and Animal Health and the Environment

Cow and sheep manure contains beneficial nutrients nitrogen (N) carbohydrates (CHO) and Organic matter (OM), and is therefore used as an organic fertilizer. However, the excessive use of manure can cause environmental problems and negatively affect human and animal health because these materials also contain large amounts of heavy metals, especially selenium (Se), nickel(Ni), cobalt(Co), tin(Sn), lead(Pb), and cadmium(Cd) To assess the potential risks due to increased concentrations of heavy metals in sheep and cow manure.The compost was collected from three different regions in Kirkuk, Diyala and Sulaymaniyah governorates in Iraq and Kurdistan Region where the results showed The content of cow and sheep manure of organic matter (OM), carbohydrates (CHO) and nitrogen (N) levels were normal, which are beneficial nutrients for soil and plants and used as organic fertilizer. The level of selenium, nickel and cobalt was higher in cow manure compared to sheep manure in all governorates. The level of lead and cadmiumwas higher in sheep manure in Diyala governorates compared to cow manure

Geranylgeranyl transferase regulates CXC chemokine formation in alveolar macrophages and neutrophil recruitment in septic lung injury

Hasan Z, Rahman M, Palani K, Syk I, Jeppsson B, Thorlacius H. Geranylgeranyl transferase regulates CXC chemokine formation in alveolar macrophages and neutrophil recruitment in septic lung injury. Am J Physiol Lung Cell Mol Physiol 304: L221-L229, 2013. First published December 14, 2012; doi:10.1152/ajplung.00199.2012.-Overwhelming accumulation of neutrophils is a significant component in septic lung damage, although the signaling mechanisms behind neutrophil infiltration in the lung remain elusive. In the present study, we hypothesized that geranylgeranylation might regulate the inflammatory response in abdominal sepsis. Male C57BL/6 mice received the geranylgeranyl transferase inhibitor, GGTI-2133, before cecal ligation and puncture (CLP). Bronchoalveolar lavage fluid and lung tissue were harvested for analysis of neutrophil infiltration, as well as edema and CXC chemokine formation. Blood was collected for analysis of Mac-1 on neutrophils and CD40L on platelets. Gene expression of CXC chemokines, tumor necrosis factor-alpha (TNF-alpha), and CCL2 chemokine was determined by quantitative RT-PCR in isolated alveolar macrophages. Administration of GGTI-2133 markedly decreased CLP-induced infiltration of neutrophils, edema, and tissue injury in the lung. CLP triggered clear-cut upregulation of Mac-1 on neutrophils. Inhibition of geranylgeranyl transferase reduced CLP-evoked upregulation of Mac-1 on neutrophils in vivo but had no effect on chemokine-induced expression of Mac-1 on isolated neutrophils in vitro. Notably, GGTI-2133 abolished CLP-induced formation of CXC chemokines, TNF-alpha, and CCL2 in alveolar macrophages in the lung. Geranylgeranyl transferase inhibition had no effect on sepsis-induced platelet shedding of CD40L. In addition, inhibition of geranylgeranyl transferase markedly decreased CXC chemokine-triggered neutrophil chemotaxis in vitro. Taken together, our findings suggest that geranylgeranyl transferase is an important regulator of CXC chemokine production and neutrophil recruitment in the lung. We conclude that inhibition of geranylgeranyl transferase might be a potent way to attenuate acute lung injury in abdominal sepsis

Rho-Kinase Signaling Regulates Pulmonary Infiltration of Neutrophils in Abdominal Sepsis via Attenuation of CXC Chemokine Formation and Mac-1 Expression on Neutrophils.

ABSTRACT: Excessive neutrophil infiltration is a major component in septic lung injury, although the signaling mechanisms behind pulmonary recruitment of neutrophils in polymicrobial sepsis remain elusive. Herein, we hypothesized that Rho-kinase activity may play a significant role in pulmonary neutrophil recruitment and tissue damage in abdominal sepsis. Male C57BL/6 mice were treated with the Rho-kinase inhibitor Y-27632 (0.5 or 5 mg/kg) before cecal ligation and puncture. Bronchoalveolar lavage fluid and lung tissue were harvested for analysis of neutrophil infiltration, as well as edema and CXC chemokine formation. Blood was collected for analysis of Mac-1 on neutrophils and CD40L on platelets as well as soluble CD40L and metalloproteinase-9 (MMP-9) in plasma. CLP triggered significant pulmonary damage characterized by neutrophil infiltration, increased levels of CXC chemokines, and edema formation in the lung. Furthermore, CLP up-regulated Mac-1 expression on neutrophils, decreased CD40L on platelets and increased soluble CD40L and MMP-9 in the circulation. Interestingly, inhibition of Rho-kinase dose-dependently decreased CLP-induced neutrophil expression of Mac-1, formation of CXC chemokines and edema as well as neutrophil infiltration and tissue damage in the lung. Moreover, Rho-kinase inhibition significantly reduced sepsis-provoked gene-expression of CXC chemokines in alveolar macrophages. In contrast, Rho-kinase inhibition had no effect on platelet shedding of CD40L or plasma levels of MMP-9 in septic mice. In conclusion, these data demonstrate that the Rho-kinase signaling pathway plays a key role in regulating pulmonary infiltration of neutrophils and tissue injury via regulation of CXC chemokine production in the lung and Mac-1 expression on neutrophils in abdominal sepsis

Rac1 signaling regulates sepsis-induced pathologic inflammation in the lung via attenuation of Mac-1 expression and CXC chemokine formation.

Excessive neutrophil recruitment is a major feature in septic lung damage although the signaling mechanisms behind pulmonary infiltration of neutrophils in sepsis remain elusive. In the present study, we hypothesized that Rac1 might play an important role in pulmonary neutrophil accumulation and tissue injury in abdominal sepsis. Male C57BL/6 mice were treated with Rac1 inhibitor NSC23766 (5 mg/kg) before cecal ligation and puncture (CLP). Bronchoalveolar lavage fluid and lung tissue were collected for the quantification of neutrophil recruitment and edema and CXC chemokine formation. Blood was collected for the determination of Mac-1 on neutrophils and proinflammatory compounds in plasma. Gene expression of CXC chemokines and tumor necrosis factor alpha was determined by quantitative reverse transcription-polymerase chain reaction in alveolar macrophages. Rac1 activity was increased in lungs from septic animals, and NSC23766 significantly decreased pulmonary activity of Rac1 induced by CLP. Administration of NSC23766 markedly reduced CLP-triggered neutrophil infiltration, edema formation, and tissue damage in the lung. Inhibition of Rac1 decreased CLP-induced neutrophil expression of Mac-1 and pulmonary formation of CXC chemokines. Moreover, NSC23766 abolished the sepsis-evoked elevation of messenger RNA levels of CXC chemokines and tumor necrosis factor alpha in alveolar macrophages. Rac1 inhibition decreased the CLP-induced increase in plasma levels of high mobility group protein B1 and interleukin 6, indicating a role of Rac1 in systemic inflammation. In conclusion, our results demonstrate that Rac1 signaling plays a key role in regulating pulmonary infiltration of neutrophils and tissue injury via regulation of chemokine production in the lung and Mac-1 expression on neutrophils in abdominal sepsis. Thus, targeting Rac1 activity might be a useful strategy to protect the lung in abdominal sepsis

Rho-kinase regulates induction of T-cell immune dysfunction in abdominal sepsis.

T-cell dysfunction increases susceptibility to infections in patients with sepsis. In the present study, we hypothesized that Rho-kinase signaling might regulate induction of T-cell dysfunction in abdominal sepsis. Male C57BL/6 mice were treated with the specific Rho-kinase inhibitor Y-27632 (5 mg/kg) prior to cecal ligation and puncture (CLP). Spleen CD4 T-cell apoptosis, proliferation and regulatory T-cells (CD4(+)CD25(+)Foxp3(+)) were determined by flow cytometry. Formation of IFN-γ and IL-4 in the spleen and plasma levels of HMBG1 and IL-6 were quantified by use of ELISA. It was found that CLP evoked apoptosis and decreased proliferation in splenic CD4 T-cells. Inhibition of Rho-kinase activity decreased apoptosis and enhanced proliferation of CD4 T-cells in septic animals. In addition, CLP-evoked induction of regulatory T-cells in the spleen was abolished by Rho-kinase inhibition. CLP reduced the levels of IFN-γ and IL-4 in the spleen. Pretreatment with Y-27632 inhibited the sepsis-induced decrease in IFN-γ but not IL-4 formation in the spleen. CLP increased plasma levels of HMGB1 by 20-fold and IL-6 by 19-fold. Inhibition of Rho-kinase decreased this CLP-evoked increase of HMGB1, IL-6 and IL-17 levels in the plasma by more than 60%, suggesting that Rho-kinase regulates systemic inflammation in sepsis. Moreover, we observed that pretreatment with Y-27632 abolished CLP-induced bacteremia. Together, our novel findings indicate that Rho-kinase is a powerful regulator of T-cell immune dysfunction in abdominal sepsis. Thus, targeting Rho-kinase signaling might be a useful strategy to improve T-cell immunity in patients with abdominal sepsis